From retina to brain

Nonsynaptic localization of the excitatory amino acid transporter 4 in photoreceptors

Objective To investigate the most practical examination method for diagnosis and treatment of retinal breaks and evaluate the value of early diagnosis and treatment for retinal breaks. Methods A total of 192 patients (216 eyes) diagnosed as posterior vitreous detachment (PVD) by ophthalmic A/B ultrasound underwent indirect ophthalmoscope, 90D mirror under the slit lamp, and three-mirror lens fundus examinations after mydriasis. Patients with retinal breaks uncombined with peripheral retinal detachment or accompanied by local superficial detachment were treated by 532 nm laser photocoagulation around the breaks; while patients with retinal breaks accompanied by peripheral retinal detachment were treated by surgery. Results In 216 eyes of PVD patients, 28 eyes of retinal tears were found by indirect ophthalmoscope, accounting for 12.96%; 26 eyes of retinal tears were found by the 90D under the slit lamp, accounting for 12.04%; 31 eyes of retinal tears were found by three-mirror lens, accounting for 14.35%, and there was no significant difference among the three groups inexamination results (P>0.05). Twenty-two eyes with retinal tears uncombined with peripheral retinal detachment underwent preventive photocoagulation treatmen for once; 6 eyes with retinal tears accompanied by local superficial detachment underwent retinal photocoagulation for 2 times; 3 eyes with retinal breaks accompanied by large range retinal detachment underwent scleral cryotherapy combined with scleral buckling. Followed up for 6 months, all the patients with retinal breaks had good therapeutic effect, without new retinal detachment. Conclusions Early detection and early diagnosis for retinal breaks are very important. The timely and proper treatment of retinal photocoagulation is the key to prevent the occurrence of retinal detachment; three-mirror lens is still very useful to detect retinal breaks. Primary level doctors should pay attention to the the importance and application of these methods. Key words: Retinal breaks; Posterior vitreous detachment; Photocoagulation; Early diagnosis; Treatment

Aims: The isolated mouse and postmortem human retina can serve as a sensitive biosensor for preclinical drug testing. To facilitate testing with minimal amounts of potential drug molecules or nanostructures, we developed a closed‐perfusion tERG setup. The major challenge with small perfusate volumes was the healthiness and stability of the retina in prolonged experiments.Methods: We conducted ex vivo tERG experiments using WT C57BL/6J and Gnat1‐/‐ mouse, and human donor retinas (Lion's Eye Bank, Utah). The isolated mouse or punches of human retinas were embedded in a specimen holder and the retina was perfused with a constant flow (5–6 ml/min) from photoreceptor side, circulated with a peristaltic pump. Added Penicillin‐Streptomycin to prevent bacterial growth. 50 μM BaCl2 inhibited the K+ currents in Müller glial cells, and 20 μΜ D, L‐2‐amino‐4‐phosphonobutyric acid was added to isolate the rod component. Stimulated the dark‐adapted retina with 530 nm full‐field flashes. The electrical connection through the perfusate from the peristaltic pump to the specimen holder was cut off with burettes that also attenuated the pulsation from the pump. The stimulus and recording devices were remotely controlled by an automatic setup implemented with LabVIEW software.Results: The minimum perfusate needed in our closed perfusate circulation was 50 ml. Pen‐Strep was indispensable for long recordings. With mice, rod responses were stable for more than 24 hours and rod and cone bipolar cell responses for up to 12 hours. Rod responses from human donor tissue were stable up to 12 hours. We used our setup to test the zwitterionic polymer poly (sulfobetaine methacrylate), serving as a promising material for thermoresponsive nanostructures, for possible harmful effects on mouse rod and bipolar cell functioning. No effects on rod and bipolar cell responses were observed.Conclusions: Our method enables long, stable tERG recordings with closed perfusion on mouse and human retinas, allowing fast preclinical drug efficacy and biocompatibility testing with tiny amounts of drug molecules, materials, or nanostructures. Sulfobetaine as monomer or linear polymer did not harm rod, ON‐bipolar, or synapse functioning, suggesting the material can safely be used for constructing thermoresponsive nanostructures for drug delivery.

The Old World macaque monkey and New World common marmoset provide fundamental models for human visual processing, yet the human ancestral lineage diverged from these monkey lineages over 25 Mya. We therefore asked whether fine-scale synaptic wiring in the nervous system is preserved across these three primate families, despite long periods of independent evolution. We applied connectomic electron microscopy to the specialized foveal retina where circuits for highest acuity and color vision reside. Synaptic motifs arising from the cone photoreceptor type sensitive to short (S) wavelengths and associated with "blue-yellow" (S-ON and S-OFF) color-coding circuitry were reconstructed. We found that distinctive circuitry arises from S cones for each of the three species. The S cones contacted neighboring L and M (long- and middle-wavelength sensitive) cones in humans, but such contacts were rare or absent in macaques and marmosets. We discovered a major S-OFF pathway in the human retina and established its absence in marmosets. Further, the S-ON and S-OFF chromatic pathways make excitatory-type synaptic contacts with L and M cone types in humans, but not in macaques or marmosets. Our results predict that early-stage chromatic signals are distinct in the human retina and imply that solving the human connectome at the nanoscale level of synaptic wiring will be critical for fully understanding the neural basis of human color vision.

The Old World macaque monkey and New World common marmoset provide fundamental models for human visual processing, yet the human ancestral lineage diverged from these monkey lineages over 25 Mya. We therefore asked whether fine-scale synaptic wiring in the nervous system is preserved across these three primate families, despite long periods of independent evolution. We applied connectomic electron microscopy to the specialized foveal retina where circuits for highest acuity and color vision reside. Synaptic motifs arising from the cone photoreceptor type sensitive to short (S) wavelengths and associated with "blue-yellow" (S-ON and S-OFF) color-coding circuitry were reconstructed. We found that distinctive circuitry arises from S cones for each of the three species. The S cones contacted neighboring L and M (long- and middle-wavelength sensitive) cones in humans, but such contacts were rare or absent in macaques and marmosets. We discovered a major S-OFF pathway in the human retina and established its absence in marmosets. Further, the S-ON and S-OFF chromatic pathways make excitatory-type synaptic contacts with L and M cone types in humans, but not in macaques or marmosets. Our results predict that early-stage chromatic signals are distinct in the human retina and imply that solving the human connectome at the nanoscale level of synaptic wiring will be critical for fully understanding the neural basis of human color vision.

l-Glutamate is the major excitatory transmitter in the vertebrate retina and plays a central role in the transmission of the various retinal neurons. Glutamate is removed from the extracellular space by at least five different glutamate transporters. The cellular distribution of these has been studied so far mainly using immunocytochemistry. In the present study non-radioactive in situ hybridisation using complementary RNA probes was applied in order to identify the cell types of rat retina and optic nerve expressing generic GLT1, GLT1 variant (GLT1v or GLT1B), GLAST and EAAC1. The results were compared with immunocytochemical data achieved using affinity-purified antibodies against transporter peptides. In the immunohistochemical studies the human retina was included. The study showed that in the rat retina GLT1v and EAAC1 were coexpressed in various cell types, i.e. photoreceptor, bipolar, horizontal, amacrine, ganglion and Müller cells, whereas GLAST was only detected in Müller cells and astrocytes. In the rat optic nerve GLT1v and EAAC1 were preferentially expressed in oligodendrocytes, whereas GLAST was revealed to be present mainly in astrocytes. Generic GLT1 could not be detected in the retina or optic nerve. The cellular distribution of glutamate transporters (only immunocytochemistry) in the human retina was very similar to that of the rat retina. Remarkable results of our studies were that generic GLT1 was not detectable in the rat (and human) retina and that GLT1v and EAAC1 were demonstrable in most cell types of the retina (including photoreceptor cells and their terminals).

Objective To investigate the effects of preventive retinal photocoagulation for low,moderate and high myopia with peripheral retinal degeneration or tear befor femtosecond laser LASIK.Methods 2000 eyes of 1000 cases with myopia,aged 18-50 years old with-1.00 ～-13.00D myopia refraction and -0.50 ～-4.00D astigmatism were enrolled in this study.The cases were divided into 3 groups according to preoperative refraction.In group Ⅰ,there were 350 cases(700 eyes) with refraction-1.00 ～-3.00D; The group Ⅱ,included 360 cases (720 eyes) with refraction-3.25 ～-6.00D ; The group Ⅲ included 290 cases(580 eyes) with refraction-6.25 ～-13.00D.All cases were examined with 90D preset lens before LASIK.Results The severe peripheral retinal degeneration and tears were found in 100 eyes,with 20 eyes in group Ⅰ,44 eyes in group Ⅱ,36 eyes in group Ⅲ,and most of which occurred in the temporal side.The rates of severe peripheral retinal degeneration and tears of group Ⅱ and Ⅲ were higher than group Ⅰ.One hundred eyes with peripheral retinal degeneration or retinal tear were treated with 532nm laser photocoagulation before LASIK.One month after laser photocoagulation,the retinal tears were sealed well and retinal condition was good.Pigment spots were obviously formed.Patients were followed up for six months to one year,and no rhegmatogenous retinal detachment was found.Conclusion Preventive 532 nm laser photocoagulation for myopia with peripheral retinal degeneration or retinal tear before LASIK is safe and effective.It can reduce or avoid the occurrence of postoperative retinal detachment to secure postoperative results. Key words: Myopia; Laser, femtosecond; LASIK; Photocoagulation, retinal, preventive

Objective To observe the effects of central retinal thickness of function among high altitude Tibetan residents with non-proliferative diabetic retinopathy. Methods Ninety cases (180 eyes) of both Tibetan plateau residents (high altitude group) and Han Chinese residents in low altitude (low altitude group) with non-proliferative diabetic retinopathy patients were selected from January 2015 to June 2016, excluding the central retina significant bleeding, oozing and significant other retinopathy. Based on the thickness of central retinal by optical coherence tomography (OCT, cirrus400 Zeiss) examination (center 1mm, 1-3mm, 3-6mm straight by range), patients from different altitudes were re-divided into two groups of normal central retinal thickness group and abnormal group with 45 patients (90) of each group. Function of central retinal thickness of the patients were observed including ERG data, angiography data, f-ERG in dark-adapted rod, the mixed reaction cones, cone response, flashing light 30Hs reaction, VEP indicators and correlation between these were observed and analyzed. Results Central retinal thickness of the normal central retinal average thickness of 276.4-304.9 um; high altitude group central retinal thickness of normal and abnormal group Max-R (dark-adapted rod maximal response and the mixed reaction cones) a amplitudes, Max-R b amplitudes, cone-R (cone response) a wave amplitude, cone-R b amplitudes, 30Hs blinking light reaction P1 wave amplitude decreased (F=3.716, 6.524, 3.825, 4.59, 7.52, P=0.03, 0.00, 0.03, 0.04, 0.00); PVEP of P100 latency (F=9.35, P=0.00), P100 amplitude was significantly lower (F=7.15, P=0.00); low altitude population centers retinal thickness normal and abnormal group Max-R (dark adaptation reaction was mixed reaction largest rod and cone cells) a amplitude value, Max-R b amplitudes, cone-R (cone response) a amplitudes, Cone-R b amplitudes, 30Hs blinking light reaction P1 wave amplitude decreased (F=3.56, 5.42, 4.25, 5.12, 6.52, P=0.03, 0.02, 0.03, 0.02, 0.00); PVEP of P100 latency (F=2.19, P=0.10), P100 amplitude was significantly lower F=9.85, P=0.00); central retinal thickness normal high altitude group Compare Max-Ra amplitudes and reduced low altitude group FERG (F=1.796, P=0.08), Max-R b wave amplitude decrease F=3.114, P=0.03), Cone-Ra amplitude value decreased (F=3.758, P=0.04), Cone-R b wave amplitude decrease (F=3.714, P=0.03), 30Hs scintillation light reaction P1 wave amplitude decrease (F=3.718, P=0.03); PVEP of P100 latency (F=2.11, P=0.10), P100 amplitude was significantly reduced (F=10.15, P=0.00). central retinal thickness abnormal high altitude and low altitude cluster group FERG Compare Max-R, a wave amplitude decrease is not obvious (F=2.792, P=0.06), Max-R b wave amplitude decrease (F=6.485, P=0.00), Cone-R a wave amplitude decrease (F=3.518, P=0.03), Cone-R b wave amplitude decrease (F=3.724, P=0.03), 30Hs scintillation light reaction P1 wave amplitude decreased (F=4.812, P=0.02); P100 latency changes PVEP not obvious (F=8.49, P=0.00), P100 wave amplitude value fell significantly (F=1.72, P=0.09). Conclusions After the Tibetan plateau residents and non-residents of low-lying Han proliferative diabetic retinopathy retinal thickness changes in the eye center, cones function has a more significant damage, central retinal morphological changes accompanied by a significant change in macular function. Key words: Diabetic retinopathy; Central retinal thickness; Central function; Clinical Observation

Objective To observe the clinical features of combined central retinal artery and vein occlusion.Methods The clinical data of eight patients of combined central retinal artery and vein occlusion diagnosed by fundus examination and fundus fluorescein angiography (FFA) was analyzed retrospectively,including the causes,fundus manifestations and FFA features.Results 4/8 patients had hypertension and dyslipidemia,2/8 patients had traumatic retrobulbar hemorrhage,one patient had orbital cellulitis and one patient had systemic lupus erythematosus.All the patients had posterior pole retinal edema,hemorrhage,thin retinal artery,dilated vein,and papilledema.FFA showed delayed arterial filling,and there was no filling of retinal arterial branches until the late stage of FFA.Laminar flow delayed in large retinal veins,and there was no filling or only retrograde filling in retinal vein branches.Large areas with dot-like or patchy weak choroidal fluorescence can be observed in five patients.Conclusions Combined central retinal artery and vein occlusion is rare with complex etiology.The fundus manifestations and FFA features are atypical,but have features of central retinal artery occlusion and central retinal vein occlusion. Key words: Retinal artery occlusion/etiology; Retinal vein occlusion/etiology; Fluorescein angiography; Disease attributes

Objective To analyze the causes of retinal detachment in patients with open ocular trauma after vitrectomy. Methods The clinical data of 129 patients with open ocular trauma underwent vitrectomy from Jul. 2013 to Jul. 2018 were retrospectively analyzed. A total of 114 patients in the non-retinal detachment group and 15 patients in the retinal detachment group were included. The cause of retinal detachment after vitrectomy for open ocular trauma were analyzed. Results There was no significant difference in gender, age or type of injury between the two groups (P>0.05). The factors of postoperative retinal detachment were related to combined with proliferative vitreoretinopathy (PVR), retinal detachment, operation time and intraoperative combined therapy. In the retinal detachment group the incidence of posperative retinal detachment in patients of PVR was higher than that in the non-retinal detachment group (P=0.002). The difference of preoperative incidence of retinal detachment (P=0.032) and later operation time (P<0.001) were statistically significant. The proportion of intraoperative scleral cryotherapy and retinotomy in the retinal detachment group (P=0.020, 0.000) was higher than that in the non-retinal detachment group during the operation. Conclusion PVR, preoperative retinal detachment, delayed surgery and over-operation during surgery are the main factors of retinal detachment after open ocular trauma surgery. Key words: Trauma, open ocular; Vitrectomy; Detachment, retinal; Vitreoretinopathy, proliferative

Objective To observe the effect of conditional knocking out (KO) vascular endothelial growth factor (VEGF) gene on the mouse model of oxygen induced retinopathy (OIR). Methods The conditional VEGF KO mice were generated using Cre-Loxp technology, resulting in the deletion of VEGF in a portion of Muller cells permanently in mouse retina. Cre positive was CKO mice, Cre negative was NKO mice. OIR was induced by keeping mice in 75% oxygen at postnatal 7 days (P7) to P12 and in room air from P12 to P17 (each 20 mice for CKO and NKO, respectively). The mice mortality was analyzed. At day P17, the percentage of retinal avascular area was calculated using retinal flat-mounting with fluorescence angiography, the number of vascular endothelial cell nucleus breaking through retinal inner limiting membrane was counted with hematoxylin eosin (HE) staining of retinal sections, and the expression of hypoxia-inducible factor-1α (HIF- 1α) was detected by immunofluorescence analysis. Results During the development of OIR, the mortality rate of CKO mice (65.00%) was higher than that of NKO mice (30.00%) with the significant difference (x2=4.912, P=0.027). At day P17, all the mice retinas were harvested. The retinal fluorescence angiography displayed that the normal retinal vascularization of CKO mice was delayed, and large avascular areas were observed. Meanwhile, rare new vascular plexus was found in CKO mice and the thickness of whole retina decreased dramatically. In contrast, NKO mice developed larger area of normal retinal vascular network structure with higher blood vessel density and more new vascular plexus with obvious fluorescein leakage. The percentage of avascular area in CKO mice [(28.31±11.15)%] was higher than NKO mice [(16.82±7.23)%] with the significant difference (t=2.734, P=0.014). The HE staining of retinal sections indicated smaller counts of vascular endothelial cell nucleus breaking through retinal inner limiting membrane in CKO mice (26.10±6.37) when compared to NKO mice (28.80±7.59), the difference was significant (t=2.437, P=0.016). The immunofluorescence analysis showed stronger expression of HIF-1α in CKO mice than NKO mice, which was mainly located in the retinal ganglion cell layer. Conclusions The local VEGF gene knockout partially inhibits retinal neovascularization in OIR mice. However, it also suppresses the normal retinal blood vascular development with a decrease of OIR mice survival ability. Key words: Microglia; Mice, knockout; Vascular endothelial growth factors; Disease models animal; Hypoxia-inducible factor 1, alpha subunit

Background Researches showed that some claudin subtypes are closely related to the integrity of blood retina barrier and participate in pathological angiogenesis.Ciaudin-23 is a novel discovered claudin subtype.Understanding its expression in the retina and its relationship with retinal vascular lesion is very important for the targeting treatment of retinal vascular diseases.Objective The aim of this study was to investigate the differential expression of claudin-23 in the retina of mice with oxygen-induced retinopathy(OIR).Methods OIR mice models were established by putting postnatal day 7 (P7)SPF C57BL/6J mice in the enclosed container with oxygen concentration of 75％ ±3％ together with mother mice for 5 days.The age-matched normal mice were used as controls.Fluorescein isothiocyanate dextran (FITC-dextran) was retro-orbitally injected to mice to confirm the models by whole-mount retina.Expressions of claudin-23 mRNA and protein in P12,P17 and P25 mice retinas were examined using real-time PCR and Western blot.lmmunofluorescent staining of frozen section was used to identify the expression and location of claudin-23 in the normal and OIR mice.Results Tortuous and engorged retinal vessels were found in P17 mice retina of the OIR group.Nonperfusion zones,abnormal capillary network and lots of pathological neovascular tufts were observed on the whole-mount retinas.The level of claudin-23 mRNA was slowly increased with the aging,reaching 2.3 fold in P25 normal mice more than P7 mice.However,that in OIR mice demonstrated 12.5-fold elevation from P7 to P25.There were no significant change in the mRNA and protein levels of claudin-23 in P7 mice retina between the normal group and OIR group(P＞0.05).But the mRNA level of claudin-23 in P12,P17 and P25 mice retinas was significantly higher in the OIR group than that in normal group(P＜0.05).The protein level of claudin-23 in P25 mice retina was significantly higher in the OIR group than that in normal group(P＜0.05).Claudin23 expressed mainly in inner nuclear cell layer and retinal ganglion cell layer of the normal mice,but claudin-23 expression was obviously increased in OIR mice.No colocalization of claudin-23 and Isolectin B4 was found in the retina in both normal and OIR mice by double immunofluorescent staining.Conclusions Claudin-23 expression upregulates in the retina of OIR mice.Claudin-23 locates in retinal neurons but not vascular endothelial cells. Key words: Claudin-23; Retina; Oxygen-induced retinopathy

Objective To evaluate the effectiveness and safety of 25G illumination aided scleral buckling surgery for treatment of rhegmatogenous retinal detachment (RRD). Methods This is a retrospective case control study. Fifty-seven RRD patients (57 eyes) were enrolled in this study. There were 35 males (35 eyes) and 22 females (22 eyes). The patients were randomly divided into ophthalmoscope group (29 patients, 29 eyes) and illumination group (28 patients, 28 eyes). There was no differences in the data of gender, age, onset time, logarithm of the minimum angle of resolution (logMAR) best corrected visual acuity(BCVA) and information of retinal tears between the two groups (P>0.050). The patients in the ophthalmoscope group received operation of conventional scleral buckling with binocular indirect ophthalmoscope. The patients in the illumination group received scleral buckling surgery with the aid of intraocular illumination and noncontact wide-angle viewing system. The follow-up was ranged from 6 to 12 months. The BCVA, intraocular pressure, fundus examination and complications were observed and recorded. Results The difference of operation time between two groups was significant (t=2.124, P=0.031). In the ophthalmoscope group, 26 eyes (89.7%) achieved retinal reattachment, 3 eyes (10.3%) failed in retinal reattachment. In the illumination group, 26 eyes (92.8%) achieved retinal reattachment, 2 eyes (7.2%) failed in retinal reattachment. There was no difference of retinal reattachment rate (P=1.000). Five eyes failed in retinal reattachment, 3 eyes received sclera buckling surgery, 2 eyes received vitrectomy with silicone oil tamponade. The final reattachment ratios were both 100%. BCVA increased in both groups compared with pre-surgery BCVA (t=4.529, 5.108; P<0.001). The difference of BCVA between two groups was not significant (t=0.559, P=0.458). There was no significant difference of intraocular pressure and complications before and after surgery in both two groups (t=−1.386, −1.437; P=0.163, 0.149). The difference of intraocular pressure between two groups was not significant (t=0.277, P=0.730). Subretinal hemorrhage occurred in 1 eye in the ophthalmoscope group. There was no iatrogenic retinal break, choroidal hemorrhage and endophthalmitis in the two groups. Conclusion 25G intraocular illumination aided buckling surgery for treatment of RRD is fast, safe and effective. Key words: Retinal detachment/surgery; Scleral buckling

Objective To observe the clinical efficacy of the modified external route surgery for retinal detachment (RD). Methods Sixty-three eyes with rhegmatogenous retinal detachment were enrolled in this study,circling were performed,and then the drainage of subretinal fluid,retinal cryopexy through sclera,and buckling were performed.The condition of retinal reattachment and the location of breaks were examined through binocular indirect ophthalmoscopy.At the end of surgery,the location of filtered silica gel was adjusted if necessary.After the surgeries,all the patients were followed up.Visual acuity,the condition of retinal reattachment,and the complications were observed each time. Results Sixty-three cases got complete reattachment.The reaction of retinal cryotherapy was seen clearly under the microscope,and the location of retinal breaks and the condition of retinal reattachment were observed more thoroughly through binocular indirect ophthalmoscopy.Conclusions The modified surgery is simple,convenient,easily observe to perform retinal reattachment surgery,and the outcome is reliable and satisfactory. Key words: Retinal detachment; Modified surgery

Objective To evaluate the effects of retinal cryotherapy and intravitreal triamcinolone injection to treat Coats' disease with exudative retinal detachment. Methods This was a prospective consecutive case study, and 21 patients (21 eyes) with Coats' disease accompanied by exudative retinal detachment were enrolled. There were 19 males and two females patients, aged from two to 18 years.Fifteen eyes had partial retinal detachment (stage 3A) and six eyes had total retinal detachment (stage 3B).All patients underwent eryocoagulation and intravitreal triamcinolone injection. Three eyes underwent sclerotomy to drain subretinal fluid. Four eyes underwent further treatment by photocoagulation or cryotherapy for the residual abnormal blood vessels after the surgery. The follow-up ranged from three to 15 months with an average of seven months. Visual acuity, intraocular pressure, eye position and eye movements, slit lamp microscope, indirect ophthalmoscope and color fundus imaging were followed up. The last follow-up time was considered as the judgment time for the therapeutic effects. Results Six eyes had increased intraocular pressure after the surgery, which was controlled by local drug treatment. At the end of follow-up, 19 eyes had reattached retina, one eye had partial retinal detachment and one eye had total retinal detachment. The vision improved in three eyes, unchanged in 14 eyes, and decreased in two eyes. Other complications included strabismus (one eye) and cataract (four eyes). Conclusion The combined treatment of cryotherapy and intravitreal triamcinolone injection is an effective therapy for the Coats' disease with exudative retinal detachment, as retina reattaches and visual function is saved in most patients after this treatment. Key words: Retinal diseases/surgery; Retinal detachment/surgery; Cryotherapy/methods; Triamcinolone acetonide/therapeutic use; Coats' disease